The present invention relates to molecules involved in cell signaling, and in particular with molecules involved in transducing a signal through a G protein coupled receptor.
The actions of many extracellular signals are mediated by the interaction of guanine nucleotide-binding regulatory proteins (G proteins) and G-protein coupled receptors (GPCRs). Individual GPCRs activate particular signal transduction pathways through binding to G proteins, which in turn transduce a signal to the cell to elicit a response from the cell. GPCRs are known to respond to numerous extracellular signals, including neurotransmitters, drugs, hormones, odorants and light. The family of GPCRs has been estimated to include several thousands members, fully more than 1.5% of all the proteins encoded in the human genome. The GPCR family members play roles in regulation of biological phenomena involving virtually every cell in the body. The sequencing of the human genome has led to identification of numerous GPCRs; although the ligands and functions of many of these GPCRs are known, a significant portion of these identified receptors are without known ligands. These latter GPCRs, known as xe2x80x9corphan receptorsxe2x80x9d, also generally have unknown physiological roles.
Many available therapeutic drugs in use today target GPCRs, as they mediate vital physiological responses, including vasodilation, heart rate, bronchodilation, endocrine secretion, and gut peristalsis. See, eg., Lefkowitz et al., Ann. Rev. Biochem. 52:159 (1983); Gilman, A. G. (1987) Annu. Rev. Biochem 56: 615-649; Hamm, H. E. (1998) JBC 273: 669-672; Ji ,T. H. (1998) JBC 273: 17229-17302. Kanakin, T. (1996) Pharmacological review, 48:413-463; Gudermann T. and Schultz, G. (1997), Annu. Rev. Neurosci., 20: 399-427. In fact, it has been estimated that more than 50% of the drugs in use clinically in humans at the present time are directed at GPCRs, including the adrenergic receptors (ARs). For example ligands to beta ARs are used in the treatment of anaphylaxis, shock, hypertension, hypotension, asthma and other conditions.
Although in general GPCRs require agonist binding for activation, agonist-independent signaling activity has been well documented in the native form of a variety of GPCRs. This spontaneous activation of GPCRs occurs, in both normal and pathological processes, where a GPCR cellular response is generated in the absence of a ligand. For example, native dopamine D1B and prostaglandin EP1b receptors have been found to possess constitutive activity (Tiberi and Caron 1994; Hasegawa et al. 1996). In addition, a number of GPCRs, for example, receptors for thyroid-stimulating hormone (Vassart et al. 1995), have mutants that exhibit agonist-independent activity and cause disease in humans. Experimentally, several single amino acid mutations have produced agonist independent activity in GPCRs. xcex22 and xcex12 adrenergic receptors, for example, mutated at single sites in the third cytoplasmic loop show constitutive activity (Ren et al. 1993; Samama et al. 1994). In another example, a truncation deletion of the carboxyl terminus in the thyrotropin releasing hormone receptor renders the receptor constitutively active (Nussenzveig et al. 1993; Matus-Leibovitch et al. 1995) and a smaller deletion in the second extracellular loop of the thrombin receptor causes constitutive activity (Nanevicz et al. 1995).
Increased spontaneous activity and/or basal activity of GPCRs can be decreased by inverse agonism of the GPCRs. Such methods are therapeutically important where diseases cause an increase in spontaneous GPCR activity, or where it is desirable to decrease the basal activity of GPCR. Thus, a technology for identifying inverse agonists of native and mutated GPCRs has important pharmaceutical applications. Furthermore, because certain constitutively active GPCRs can be tumorigenic, the identification of inverse agonists for these GPCRs can lead to the development of anti-tumor and/or anti-cell proliferation drugs. These compounds have become increasingly important, especially for the treatment of psychological disorders such as depression and bipolar disorder. Unfortunately, conventional assays are not particularly suited to reliably identify inverse agonists, as the activity of the GPCRs in response to an inverse agonist cannot be directly measured.
Since GPCRs and G protein signaling pathways are critical targets for therapeutics, there is a need in the art for fast, effective and reproducible methods for identifying agonists, antagonists and inverse agonists that modulate G protein signaling, and in particular compounds that regulate this signaling through a GPCR. The present invention addresses this need.
The present invention provides modified G protein xcex1-subunits which are characterized by 1) constitutive localization to the plasma membrane; 2) enhanced binding to one or more of the normal receptor binding partners for that xcex1-subunit; and 3) efficient binding to and activation of G protein binding partners. The distribution of these modified xcex1-subunits, which are tethered to the plasma membrane, allows the regulation of receptor-G protein coupling, and thus G-protein signaling, in various biological systems. Also encompassed in the present invention are nucleic acids encoding such modified G protein xcex1-subunits, and expression vectors containing such nucleic acids.
In particular, these modified G protein xcex1-subunits allow for enhanced activation of downstream signaling partners of G proteins, and thus can provide for assays having increased specificity. Thus, in one embodiment, the invention provides assays for identifying agonists, antagonists, and/or inverse agonists using a system comprising the modified G protein xcex1-subunits of the invention. The tethered xcex1-subunits provided in the assay improve the response of the GPCR to its ligand, and thus these assays are useful in identifying agonists, antagonists, and/or inverse agonists that activate a GPCR. The assays comprise contacting a membrane or cell comprising tethered xcex1-subunits with a ligand and determining the activation of the GPCR via stimulation of a downstream binding partner. The downstream binding partner used in the assay will depend upon the transduction pathway for a particular class of G protein. For example, if a tethered Gsxcex1 is used in the assay, the assay may measure levels of AC stimulation. Such assays can be whole cell assays, and preferably are membrane assays.
In another embodiment, the invention provides methods for identifying the presence of a chemical that acts as a ligand for a GPCR via an assay that directly measures G protein signaling through measurement of activation of a G protein binding partner. The ligands can be identified in an assay whereby a sample suspected of containing the chemical is contacted with either whole cells or membranes comprising tethered G protein xcex1-subunits. The presence of the ligands can be identified by measuring G protein activation of a signaling partner, e.g., AC. For example, the assay can be used to identify the presence of a drug such as an opioid in a sample, as the opioid binds to and activates the opioid receptor, which is a GPCR.
In yet another embodiment, the invention provides methods for determining the ligand for an orphan GPCR via an assay that directly measures G protein signaling through measurement of activation of downstream binding partner. The ligands can be identified using an assay with either whole cells or membranes comprising tethered G protein xcex1-subunits. The ligands can be identified by measuring G protein activation of a signaling partner, e.g., AC.
In yet another embodiment, the invention provides assays for identifying the G proteins involved in the signaling of a specific GPCR using an assay comprising membranes with different tethered G protein xcex1-subunits. By assaying the activity in membranes coexpressing the GPCR and different tethered xcex1-subunits, and measuring the activation of the G protein involved in specific G protein signaling responses, the specific G protein involved in specific GPCR signaling events can be determined.
One object of the present invention is to develop rapid and sensitive bioassays for evaluating new agonists, antagonists and/or inverse agonists for GPCRs.
Another object of the present invention is to develop a strategy for identifying ligands for GPCRs.
Yet another object of the present invention is to develop a strategy for identifying GPCRs involved in different biological processes, including disease.
Yet another object of the invention is to identify the presence of a particular ligand in a sample, e.g., the presence of a drug such as an opioid.
An advantage of the invention is that the assays can be performed using membranes, which increases both the ease of performing the assay and the efficacy of the assay.
Another advantage is that assays of the invention allow high throughput screening of GPCR activity.
Yet another advantage of the invention is that the assays of the invention directly measure GPCR activity, and thus are less labor-intensive than conventional methods for determining GPCR activity.
Yet another advantage of the invention is that the modified G protein xcex1-subunits can be epitope tagged. This provides a direct method for detecting these proteins, as well as providing methods for affinity purification.
Yet another advantage is that the modified G protein xcex1-subunits can be designed to have a protease site between the active portion of the subunit and the membrane tether. This allows for isolation of the subunit based on the membrane tether, and subsequent removal of the membrane tether.
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the proteins and assays as more fully described below.